A selectable all-in-one CRISPR prime editing piggyBac transposon allows for highly efficient gene editing in human cell lines

Publikation: Beitrag in FachzeitschriftForschungsartikelBeigetragenBegutachtung

Beitragende

  • Reto Eggenschwiler - , Leibniz Universität Hannover (LUH) (Autor:in)
  • Thomas Gschwendtberger - , Leibniz Universität Hannover (LUH) (Autor:in)
  • Christian Felski - , Leibniz Universität Hannover (LUH) (Autor:in)
  • Christopher Jahn - , Leibniz Universität Hannover (LUH) (Autor:in)
  • Florian Langer - , Leibniz Universität Hannover (LUH) (Autor:in)
  • Jared Sterneckert - , Professur für iPS Zellen und neurodegenerative Erkrankungen (Autor:in)
  • Andreas Hermann - , Universität Rostock, Deutsches Zentrum für Neurodegenerative Erkrankungen e.V. (DZNE) (Autor:in)
  • Jonathan Lühmann - , Leibniz Universität Hannover (LUH) (Autor:in)
  • Doris Steinemann - , Leibniz Universität Hannover (LUH) (Autor:in)
  • Alexandra Haase - , Leibniz Universität Hannover (LUH), Deutsche Zentrum für Lungenforschung (DZL) (Autor:in)
  • Ulrich Martin - , Leibniz Universität Hannover (LUH), Deutsche Zentrum für Lungenforschung (DZL) (Autor:in)
  • Susanne Petri - , Leibniz Universität Hannover (LUH) (Autor:in)
  • Tobias Cantz - , Leibniz Universität Hannover (LUH), Max Planck Institute for Molecular Biomedicine (Autor:in)

Abstract

CRISPR prime-editors are emergent tools for genome editing and offer a versatile alternative approach to HDR-based genome engineering or DNA base-editors. However, sufficient prime-editor expression levels and availability of optimized transfection protocols may affect editing efficiencies, especially in hard-to-transfect cells like hiPSC. Here, we show that piggyBac prime-editing (PB-PE) allows for sustained expression of prime-editors. We demonstrate proof-of-concept for PB-PE in a newly designed lentiviral traffic light reporter, which allows for estimation of gene correction and defective editing resulting in indels, based on expression of two different fluorophores. PB-PE can prime-edit more than 50% of hiPSC cells after antibiotic selection. We also show that improper design of pegRNA cannot simply be overcome by extended expression, but PB-PE allows for estimation of effectiveness of selected pegRNAs after few days of cultivation time. Finally, we implemented PB-PE for efficient editing of an amyotrophic lateral sclerosis-associated mutation in the SOD1-gene of patient-derived hiPSC. Progress of genome editing can be monitored by Sanger-sequencing, whereas PB-PE vectors can be removed after editing and excised cells can be enriched by fialuridine selection. Together, we present an efficient prime-editing toolbox, which can be robustly used in a variety of cell lines even when non-optimized transfection-protocols are applied.

Details

OriginalspracheEnglisch
Aufsatznummer22154
FachzeitschriftScientific reports
Jahrgang11
Ausgabenummer1
PublikationsstatusVeröffentlicht - 12 Nov. 2021
Peer-Review-StatusJa

Externe IDs

PubMed 34773059
ORCID /0000-0002-7688-3124/work/142250009

Schlagworte